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. 1997 Jun;71(6):4385–4394. doi: 10.1128/jvi.71.6.4385-4394.1997

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

R S Weiss 1, M O Gold 1, H Vogel 1, R T Javier 1
PMCID: PMC191656  PMID: 9151828

Abstract

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.

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Selected References

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  1. Ankerst J., Jonsson N. Adenovirus type 9-induced tumorigenesis in the rat mammary gland related to sex hormonal state. J Natl Cancer Inst. 1989 Feb 15;81(4):294–298. doi: 10.1093/jnci/81.4.294. [DOI] [PubMed] [Google Scholar]
  2. Ankerst J., Jonsson N., Kjellén L., Norrby E., Sjögren H. O. Induction of mammary fibroadenomas in rats by adenovirus type 9. Int J Cancer. 1974 Mar 15;13(3):286–290. doi: 10.1002/ijc.2910130303. [DOI] [PubMed] [Google Scholar]
  3. Cedergren-Zeppezauer E. S., Larsson G., Nyman P. O., Dauter Z., Wilson K. S. Crystal structure of a dUTPase. Nature. 1992 Feb 20;355(6362):740–743. doi: 10.1038/355740a0. [DOI] [PubMed] [Google Scholar]
  4. Chu R., Lin Y., Rao M. S., Reddy J. K. Cloning and identification of rat deoxyuridine triphosphatase as an inhibitor of peroxisome proliferator-activated receptor alpha. J Biol Chem. 1996 Nov 1;271(44):27670–27676. doi: 10.1074/jbc.271.44.27670. [DOI] [PubMed] [Google Scholar]
  5. Fanning E. Simian virus 40 large T antigen: the puzzle, the pieces, and the emerging picture. J Virol. 1992 Mar;66(3):1289–1293. doi: 10.1128/jvi.66.3.1289-1293.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fisher D. E. Apoptosis in cancer therapy: crossing the threshold. Cell. 1994 Aug 26;78(4):539–542. doi: 10.1016/0092-8674(94)90518-5. [DOI] [PubMed] [Google Scholar]
  7. Fisher P. B., Babiss L. E., Weinstein I. B., Ginsberg H. S. Analysis of type 5 adenovirus transformation with a cloned rat embryo cell line (CREF). Proc Natl Acad Sci U S A. 1982 Jun;79(11):3527–3531. doi: 10.1073/pnas.79.11.3527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Freedman V. H., Shin S. I. Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell. 1974 Dec;3(4):355–359. doi: 10.1016/0092-8674(74)90050-6. [DOI] [PubMed] [Google Scholar]
  9. Green D. R., Martin S. J. The killer and the executioner: how apoptosis controls malignancy. Curr Opin Immunol. 1995 Oct;7(5):694–703. doi: 10.1016/0952-7915(95)80079-4. [DOI] [PubMed] [Google Scholar]
  10. Hinds P. W., Finlay C. A., Quartin R. S., Baker S. J., Fearon E. R., Vogelstein B., Levine A. J. Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth Differ. 1990 Dec;1(12):571–580. [PubMed] [Google Scholar]
  11. Javier R. T. Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats. J Virol. 1994 Jun;68(6):3917–3924. doi: 10.1128/jvi.68.6.3917-3924.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Javier R., Raska K., Jr, Macdonald G. J., Shenk T. Human adenovirus type 9-induced rat mammary tumors. J Virol. 1991 Jun;65(6):3192–3202. doi: 10.1128/jvi.65.6.3192-3202.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Javier R., Raska K., Jr, Shenk T. Requirement for the adenovirus type 9 E4 region in production of mammary tumors. Science. 1992 Aug 28;257(5074):1267–1271. doi: 10.1126/science.1519063. [DOI] [PubMed] [Google Scholar]
  14. Javier R., Shenk T. Mammary tumors induced by human adenovirus type 9: a role for the viral early region 4 gene. Breast Cancer Res Treat. 1996;39(1):57–67. doi: 10.1007/BF01806078. [DOI] [PubMed] [Google Scholar]
  15. Kiefer F., Courtneidge S. A., Wagner E. F. Oncogenic properties of the middle T antigens of polyomaviruses. Adv Cancer Res. 1994;64:125–157. doi: 10.1016/s0065-230x(08)60837-4. [DOI] [PubMed] [Google Scholar]
  16. Larsson G., Svensson L. A., Nyman P. O. Crystal structure of the Escherichia coli dUTPase in complex with a substrate analogue (dUDP). Nat Struct Biol. 1996 Jun;3(6):532–538. doi: 10.1038/nsb0696-532. [DOI] [PubMed] [Google Scholar]
  17. Moran E., Mathews M. B. Multiple functional domains in the adenovirus E1A gene. Cell. 1987 Jan 30;48(2):177–178. doi: 10.1016/0092-8674(87)90418-1. [DOI] [PubMed] [Google Scholar]
  18. Nilson L. A., Gottlieb R. L., Polack G. W., DiMaio D. Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous beta receptor for platelet-derived growth factor in mouse C127 cells. J Virol. 1995 Sep;69(9):5869–5874. doi: 10.1128/jvi.69.9.5869-5874.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pipas J. M. Common and unique features of T antigens encoded by the polyomavirus group. J Virol. 1992 Jul;66(7):3979–3985. doi: 10.1128/jvi.66.7.3979-3985.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shin S. I., Freedman V. H., Risser R., Pollack R. Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4435–4439. doi: 10.1073/pnas.72.11.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weiss R. S., Lee S. S., Prasad B. V., Javier R. T. Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes. J Virol. 1997 Mar;71(3):1857–1870. doi: 10.1128/jvi.71.3.1857-1870.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weiss R. S., McArthur M. J., Javier R. T. Human adenovirus type 9 E4 open reading frame 1 encodes a cytoplasmic transforming protein capable of increasing the oncogenicity of CREF cells. J Virol. 1996 Feb;70(2):862–872. doi: 10.1128/jvi.70.2.862-872.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wilkinson D., de Vries R. R., Madrigal J. A., Lock C. B., Morgenstern J. P., Trowsdale J., Altmann D. M. Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients. J Exp Med. 1988 Apr 1;167(4):1442–1458. doi: 10.1084/jem.167.4.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zhou Y. H., Zhang X. P., Ebright R. H. Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res. 1991 Nov 11;19(21):6052–6052. doi: 10.1093/nar/19.21.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]

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